Non-small cell lung carcinoma (NSCLC) is the most common cause of cancer death in the United States. The presence of metastatic disease in the mediastinal lymph nodes of NSCLC patients has profound prognostic and therapeutic implications. For instance, patients with documented disease in mediastinal lymph nodes are typically not candidates for surgical treatment, and are often treated with a combination of chemotherapy and radiotherapy. Current methods for detection of disease in mediastinal lymph nodes either lack sensitivity (computed tomography, positron emission tomography), or are invasive requiring general anesthesia (mediastinoscopy, thoracoscopy). We believe that endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) of mediastinal lymph nodes in combination with real-time RT-PCR has the potential to dramatically improve lung cancer staging. One limitation of EUS-FNA is that it currently relies on cytologic analysis of the specimen. Such an analysis is dependent on a skilled cytopathologist and lacks sensitivity. The identification of genes overexpressed in lung cancer combined with recent advances in molecular biology provides the opportunity to establish a more sensitive, and specific way to analyze EUS-FNA samples. In Section 6c. Preliminary Studies, we present preliminary data that real-time RT-PCR provides the ability to precisely determine the expression levels of lung cancer-associated mRNA, facilitating the sensitive detection of NSCLC. Furthermore, we present evidence of lung cancer-associated gene overexpression in EUS-FNA samples from patients with presumed NSCLC. The hypothesis of the proposed research is that real-time RT-PCR detection of lung cancer cells in EUS-FNA specimens of mediastinal lymph nodes in NSCLC patients is associated with clinical outcome. The successful development and validation of such a molecular diagnostic assay is likely to have a significant clinical impact. In the R21 component of this grant application we will analyze a preliminary cohort of patients and controls so that criteria for the interpretation of test results can be established. In the R33 component of the grant application these criteria will be prospectively evaluated and correlated with clinical outcome in a second, larger cohort of patients.